PROJECT SUMMARY: A central scientific question of this competing renewal is the mechanistic understanding of the inflammatory responses to cytosolic DNA. To achieve our goals, we continue using the forward genetic analysis in the wild- derived mice of the MOLF strain that diverged from classical inbred (C57BL6) mice about 1 million years ago. A phenotype that provides preliminary data for this application is the defective IFN production in MOLF macrophages in response to DNA pathogens or cytosolic DNA - a condition conferred by a hypomorphic allele of Sting. In spite of the failure to produce IFN, activated MOLF macrophages overproduce IL-6. Other wild- derived strains exhibit similar skewing of responses to DNA-viruses and DNA. Based on these data, we hypothesized that in order to avoid IFN-mediated excessive inflammation, DNA sensing pathways must have evolved to complement IFN production, perhaps with less dangerous substitutes such as IL-6. Accordingly, we will determine how wild derived mice respond to cytosolic DNA with low IFN but high IL-6--a line of investigation that will ultimately identify genes (loci) responsible for IL-6 overproduction. In the first Aim, we will test the hypothesis that retention of STING in the ER is the mechanism of switching the DNA-responses from IFN to IL-6 production. Second, we will examine the potential contribution of other DNA-sensors and pathways into STING-mediated DNA-responses in MOLF. Finally, we will use STING congenic (B6.StingMOLF/MOLF) mice, which are completely non-responsive to DNA to genetically map and identify gene(s) that confer overproduction of IL-6 in MOLF in response to DNA. In extension of genetic analysis of the trait, we will study the responses to DNA in DNAse2-/- Sting MOLF/- mice. These mice are rescued from DNAse2-/- -associated embryonic lethality with the hypomorphic allele of Sting despite high levels of inflammatory cytokines in the blood and embryonic lethality of the DNAse2-/- Sting MOLF/- females. These data suggest that some uncharacterized STING-mediated signaling exists in these mice, which will be investigated in Aim 2 of the proposal. First, we will identify the DNA-responsive cells, and use single cell RNA-sequencing analysis in these cells to identify genes with expression levels associated with the inflammatory signature and DNA- responses. Despite being confined to eQTL (expression Quantitative Trait Loci), the single cell association studies in Aim 2 will potentially reveal all associations between the phenotype and the genes, some of which will not necessarily be mapped in Aim 1 and could be completely novel. Finally, based on embryonic lethality of the DNAse2-/- Sting MOLF/- females, we will investigate potential contributions of the X-linked Tlr7 and Tlr8 into STING-mediated responses. By investigating the mechanism of DNA responses in MOLF, we hope to provide better insight into the diversity of pathologies present in human patients with interferonopathies. !